Multiple roles of Rbx1 in the VBC-Cul2 ubiquitin ligase complex
Multiple roles of Rbx1 in the VBC-Cul2 ubiquitin ligase complex
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DOI：
10.1111/j.1365-2443.2005.00869.x
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发表时间：
2005-07-01
期刊：
影响因子：
2.1
通讯作者：
Iwai, K
中科院分区：
文献类型：
--
作者：
Megumi, Y;Miyauchi, Y;Iwai, K
泛素系统的重要性很大程度上取决于泛素连接酶 E3，因为它们决定了系统的特异性。 Rbx1/ROC1/Hrt1 是一种环指蛋白，是含 cullin 的 SCF 和 VBC-Cul2 连接酶的重要组成部分。 NEDD8 对 cullin 的修饰（NEDDylation）已被证明对于 SCF 和 VBC-Cul2 的 E3 活性至关重要，并且有人建议 Rbx1 充当 cullin NEDDylation 的 E3。无名指由八个与锌离子结合的半胱氨酸和组氨酸残基组成。 Rbx1 是一种进化上高度保守的蛋白质；然而，其环指中的第八个配位残基是天冬氨酸（D97）而不是半胱氨酸。用其他 19 个氨基酸中的每一个替换 D97 表明天冬氨酸在 cullin NEDDylation 中优于半胱氨酸。然而有趣的是，不同的 D97 突变体对测试的 6 种库林表现出不同的活性。重要的是，我们能够区分 Rbx1 的 NEDDylating 活性及其在 VBC-Cul2 背景下参与的泛素化反应。此外，虽然 Rbx1 不参与控制 SCF 的稳定性，但 Rbx1 突变体会破坏 VBC-Cul2 的稳定性。总而言之，这些结果表明多种机制调节基于 cullin 的连接酶的活性和稳定性。
The importance of the ubiquitin system largely depends on ubiquitin ligases, E3s, as they determine the specificity of the system. Rbx1/ROC1/Hrt1, a RING finger protein, functions as an important component of the cullin-containing SCF and VBC-Cul2 ligases. Modification of cullins by NEDD8 (NEDDylation), has been shown to be essential for the E3 activity of both SCF and VBC-Cul2, and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation. RING finger is composed of eight cysteine and histidine residues that bind to zinc ions. Rbx1 is a highly evolutionarily conserved protein; however, the eighth coordination residue in its RING finger is aspartate (D97) rather than cysteine. Substitution of D97 with each of the other 19 amino acids demonstrates that aspartate is superior to cysteine in cullin NEDDylation. Interestingly, however, different D97 mutants demonstrate different activities towards 6 cullins tested. Importantly, we were able to discriminate between the NEDDylating activity of Rbx1 and its involvement in the ubiquitylation reaction within the context of VBC-Cul2. Moreover, while Rbx1 is not involved in governing the stability of SCF, Rbx1 mutants destabilize VBC-Cul2. Taken together, these results indicate that various mechanisms regulate both the activities and the stability of cullin-based ligases.